Method of imparting disease resistance to plants by reducing polyphenol oxidase activities

ABSTRACT

The present invention relates to a method for enhancing a plant&#39;s resistance to diseases and/or bruising by transforming the plant with sense and antisense polyphenol oxidase encoding sequences from potato. The invention also relates to transgenic plants and progeny thereof with reduced polyphenol oxidase activity.

This application claims the benefit of U.S. Provisional Application No.60/245,876, filed Nov. 3, 2000.

BACKGROUND OF THE INVENTION

This invention is in the field of transformed plants, particularlypotatoes, which have been rendered resistant to disease, including thevery destructive disease known as late blight, caused by Phytophthorainfestans.

One of the agronomically most important diseases is caused by the fungalpathogen P. infestans. In potato it causes late blight disease. Lateblight epidemics have caused a persistent threat to potato growers sincethe Irish Famine in the early 1800s, and late blight has re-emerged as adevastating disease in the United States with the recent establishmentof a new clonal lineage of P. infestans, designated A2 isolate US-8.During the mid 1990s, this unusually aggressive lineage replaced anearlier predominant lineage within only two years, and has caused severeepidemics since then, resulting in annual potato losses exceeding 100million dollars. There are currently no cost-effective means of US-8control because none of the commercially-available cultivars in theUnited States contain disease resistance (R-) genes against thispathogen, which is also resistant to the fungicide metalaxyl.

The lack of effective R-genes in cultivated potato is due, in part, tothe absence of R-gene breeding programs. Such efforts were discouragedby the fact that eleven R-genes from the resistant wild potato speciesSolanum demissum that had been introgressed into potato in the 1960sresulted in temporary control of late blight only (Landeo et al., In:Phytophthora infestans. Ed. Dowley L J et al., Bole Press Ltd. Dublin,Ireland, 268–274, 1995). Apparently, the agricultural use of R-genes forlate blight control results in the establishment of races that are notrecognized by R-genes, through rapid shifts in population dynamics of P.infestans.

An important biotechnology strategy to enhance disease resistance inplants is based on the identification and expression of antifungalproteins (AFP's). Reported AFP classes include defensins and other smallcysteine-rich peptides, 2S albumins, chitin-binding proteins, lipidtransfer proteins, and hydrogen peroxide-generating enzymes(Garcia-Olmedo et al., Biopolymers 47: 479–491, 1998). Unfortunately,the constitutive overexpression of AFP's in transgenic plants has notyet resulted in commercially relevant levels of late blight diseasecontrol. Thus, none of the conventional breeding and biotechnologyapproaches have resulted in the generation of potato cultivarsdisplaying durable late blight resistance.

It is well established that the enzyme polyphenol oxidase (PPO) is theenzyme which catalyzes the conversion of phenolic substrates,predominantly tyrosine, to melanin in many plant species. PPO is themajor cause of enzymatic browning in higher plant tissues, includingthat of potato. Polyphenol oxidases are plastid membrane-associated,copper metalloproteins which catalyze the hydroxylation of monophenolsto o-diphenols, and the dehydrogenation of o-diphenols to o-diquinonesin the presence of oxygen. The quinone products undergo a series ofnonenzymatic secondary autooxidation reactions to produce highlyreactive electrophiles which form melanin, as well as covalentlycrosslink with amine groups of cellular proteins, resulting in brown andblack pigment production (Newman, et al., Plant Mol. Biol. 21:1035–1051,1993; Thygesen, et al., Plant Physiol. 109:525–531, 1995). PPO is alsopresent in non-photosynthetic tissues, and in potato tubers PPO isassociated with amyloplasts of tuber cells. In potato tubers, theprimary phenolic substrate for PPO is tyrosine, which exists at highlevels in the free amino acid pool. PPO utilizes organic acids such aschlorogenic acid and caffeic acid much more rapidly than tyrosine, butthese substrates exist in potato tubers at significantly lower levelsthan tyrosine and are therefore not the primary substrates for PPO inthe tuber. PPO catalyzes the slow conversion of tyrosine todihydroxyphenyl-alanine (DOPA), and rapid conversion of DOPA to DOPAquinone, which autooxidizes to form brown and black melaninpigmentation. Enzymatic browning mediated by PPO occurs when tubertissue is damaged, usually by physical impact or long-term pressure, andloss of intracellular compartmentalization results, thereby allowing PPOto come into contact with tyrosine. In damaged tissue regions with darkmelanin formation, commonly referred to as black spot bruises, the cellwalls do not need to be broken, only disruption of intracellularmembrane integrity is required (Craft, Am. Pot. J. 43: 112–121, 1966;Stark et al., Am. Pot. J. 62: 657–666, 1985; Corsini et al., Am. Pot. J.69: 423–434, 1992).

In potato tubers, it is the action of the PPO enzyme which leads to theformation of black spot bruises after physical impact or damage to tubertissue. It is theorized that the reduced expression of PPO, throughtransformation with a DNA construct in antisense orientation or throughtransformation and cosuppression, use of a double-stranded mRNA (dsRNA)construct, the simultaneous expression of both sense and antisense RNA,or other effective means of reduced expression of PPO in potatoes willresult in reduced susceptibility of tubers to exhibit black spot bruises(see PCT Patent Applications WO 93/02195, 93/15599, and 94/03607).

It has been previously thought that PPO played a role in the plant'sinnate resistance to disease and therefore that reduced expression ofPPO would render the plant more susceptible to disease (see for exampleWO 93/15599, page 4). However, it has surprisingly been discovered inthe present invention that the opposite result is possible, that is,reduced expression of PPO can render a plant more resistant to disease.

SUMMARY

The present invention provides a method for reducing symptoms of diseasein plants, which are exposed to disease-causing organisms, by reducingexpression of polyphenol oxidase (PPO) to a level sufficient to resultin plants which have reduced levels of damage from said disease. Inpotatoes it is desirable that the PPO level in tubers be reduced. It ispreferred that the level of PPO be reduced at least 50 percent, ascompared to the same plant that has not been transformed for reducedexpression of polyphenol oxidase. It is more preferred that the level ofPPO be reduced at least 65 percent. It is more preferred that the levelof PPO be reduced at least 70 percent. It is more preferred that thelevel of PPO be reduced at least 75 percent. It is more preferred thatthe level of PPO be reduced at least 80 percent. It is more preferredthat the level of PPO be reduced at least 85 percent. It is morepreferred that the level of PPO be reduced at least 90 percent. It ismore preferred that the level of PPO be reduced at least 95 percent. Andmore preferably that the level of PPO be reduced at least 99 percent ascompared to the same plant that has not been transformed for reducedexpression of polyphenol oxidase. The reduction in expression can beeffected in a number of ways including (1) antisense transgenicconstructs for a native gene or DNA sequence for PPO, (2) cosuppressiontransgenic constructs for a native gene or DNA sequence for PPO usingsense orientation of that gene or sequence of a mutant thereof whichproduces only a non-catalytic protein, (3) transgenic constructsintended to cause a double-stranded mRNA for a PPO DNA sequence asdescribed below, and (4) the simultaneous expression of both sense andantisense mRNA for a PPO DNA sequence.

One exemplary method used in the present invention was the antisenseapproach, but the invention is not limited to the antisense method inorder to reduce expression of PPO. The tubers of the transgenic potatoplant produced following the antisense method display reduced visualsymptoms of the fungal disease-causing organism P. infestans. Thesetubers may also display enhanced disease resistance against certainother fungal pathogens that infect potato tubers. Other potato fungalpathogens include, but are not limited to, Spongospora (powdery scab),Rhizoctonia (black scurf), Fusarium(dry rot), Verticillium (Verticilliumwilt), Alternaria (early blight), Polyscytalum (skin spot), Sclerotium(white mold), Rosellinia (black rot), Helicobasidium (violet root rot),Macrophomina (charcoal rot), and Helminthosporium (silver scurf), andthe like.

The PPO levels may also be reduced in other plant species following themethodologies described in the present invention. Such other plants willthen have enhanced resistance to fungal pathogens. These fungalpathogens may include, but are not limited to, Cercospora sp. andMonilinia sp.

In another embodiment of the present invention transgenic potatoes areproduced that display an improved anti-bruising trait by employingspecific promoters. PPO is the major cause of enzymatic browning inhigher plants including potatoes. The formation of black spot bruises isthe action of this enzyme in tubers after they are physically damaged.By using the antisense approach to reduce expression of this enzyme to avery low level, the black spot bruises can be greatly limited. As aresult, tubers produced with this invention have an excellentanti-bruising trait over tubers that do not have this trait. Thepromoters used in the present invention may include a TFM7 promoter fromtomato fruits, a Sporamin A (SpoA) promoter from sweet potatoes and anADP-glucose pyrophosphorylase small subunit (smADPGPP) promoter frompotatoes.

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims and accompanying figures where:

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plasmid map for representation of pMON 21624.

FIG. 2 shows a plasmid map for representation of pMON 21652.

FIG. 3 shows a plasmid map for representation of pMON 21656.

FIG. 4 shows a plasmid map for representation of pMON 38914.

FIG. 5 shows a plasmid map for representation of pMON 38293.

DESCRIPTION OF SEQUENCES

-   1. SEQ ID NO:1 represents the sequence of a polyphenol oxidase DNA    sequence cloned from potato tuber tissues of the Ranger Russet    variety of Solanum tuberosum.-   2. SEQ ID NO:2 represents the antisense sequence of a polyphenol    oxidase DNA sequence cloned from potato tuber tissues of the Ranger    Russet variety of Solanum tuberosum.-   3. SEQ ID NO:3 shows a predicted amino acid sequence of the    polyphenol oxidase DNA sequence as shown in SEQ ID NO:1 cloned from    potato tuber tissues of the Ranger Russet variety of Solanum    tuberosum.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In order to provide a clear and consistent understanding of thespecification and the claims, including the scope given to such terms,the following definitions are provided.

“Antisense technology” means a method to introduce into cells a RNA orsingle-stranded DNA molecule that is complementary to the mRNA of atarget DNA sequence. This antisense molecule may work by forming abase-pair with the endogenous mRNA, preventing translation of the mRNAinto protein.

“Coding sequence” means a region of continuous sequential nucleic acidtriplets encoding a protein, polypeptide, or peptide sequence.

“Disease resistance” means the ability of plants to develop fewerdisease symptoms following exposure to a plant pathogen than asusceptible plant that does not exhibit disease resistance. Diseaseresistance includes complete resistance to the disease and also varyingdegrees of resistance manifested as decreased symptoms, longer survivalor other disease parameters, such as higher yield.

“Down regulation” means the reduction in expression or activity of anendogenous plant protein, usually an enzyme by various means, primarilyby use of an introduced segment of nucleic acids.

“Encoding DNA” means chromosomal DNA, plasmid DNA, cDNA, or syntheticDNA that encodes any of the enzymes disclosed herein.

“Expression” means the combination of intracellular processes, includingtranscription and translation, undergone by a coding DNA sequence suchas a structural gene to produce a polypeptide.

“Genome” as it applies to bacteria encompasses DNA of both thechromosome and plasmids within a bacterial host cell. Encoding DNAsequences of the present invention introduced into bacterial host cellscan therefore be either chromosomally integrated or plasmid-localized.The term “genome” as it applies to plant cells encompasses chromosomalDNA found within the nucleus, and organelle DNA found within subcellularcomponents of the cell. DNA sequences of the present inventionintroduced into plant cells can therefore be either chromosomallyintegrated or organelle-localized or combinations thereof.

“Homolog” is 70% or more in sequence identity. Significant homology of asequence very closely related to a probe sequence refers to thesequences hybridizing to the probe at 68° C. (at least 16 hours) andwashed at stringent conditions (68° C., final wash with 0.1×SSC/0.1%SDS). Final wash in 2×SSC at 50° C. allows identification of sequenceswith about 75% homology to the probe. However, the exact relationshipbetween stringency and sequence homology depends on base composition,the length of the probe, and the length of the homologous regions (Hamesand Higgins, 1985). Preferably the hybridization conditions refer tohybridization in which the TM value is between 35° C. and 45° C. Mostpreferably significant homology refers to a DNA sequence that hybridizeswith the reference sequence under stringent conditions.

“Hybridization” means the ability of a strand of nucleic acid to joinwith a complementary strand via base pairing. Hybridization occurs whencomplementary sequences in the two nucleic acid strands bind to oneanother.

“Identical” nucleotide or protein sequences are determined by usingprograms such as GAP or BestFit from GCG (Genetics Computer Group, Inc.,Madison, Wis.) using the default parameters.

“Overexpression” means the expression of a polypeptide or proteinencoded by a DNA sequence introduced into a host cell, wherein saidpolypeptide or protein is either not normally present in the host cell,or wherein said polypeptide or protein is present in said host cell at ahigher level than without the introduced sequence during at least onepart of the plant's life cycle.

“Plant” is used herein in a broad sense and means differentiated plantsas well as undifferentiated plant material, such as protoplasts, plantcells, seeds, plantlets etc., that under appropriate conditions candevelop into mature plants, the progeny thereof, and parts thereof suchas cuttings, leaves, flowers, fruits of such plants.

“Polyadenylation signal” or “polyA signal” means a nucleic acid sequencelocated 3′ to a coding region that promotes the addition of adenylatenucleotides to the 3′ end of the transcribed mRNA from the codingregion.

“Promoter” or “promoter region” means a nucleic acid sequence, usuallyfound upstream (5′) to a coding sequence, that controls expression ofthe coding sequence by controlling production of messenger RNA (mRNA) byproviding the recognition site for RNA polymerase or other factorsnecessary for start of transcription at the correct site.

“Regeneration” means the process of growing a plant from a plant cell(e.g., plant protoplast or explant).

“Resistance gene” is a nucleic acid sequence encoding a protein that isdirectly or indirectly involved in the induction of a signaltransduction pathway eventually leading to a plant defense responseagainst any pathogen or insect, upon contact or infective exposure ofthe plant with that particular pathogen or insect. Resistance geneproducts are expressed in response to pathogen signal molecules termedelicitors.

“Selectable marker” means a nucleic acid sequence whose expressionconfers a phenotype facilitating identification of cells containing theselectable marker. Selectable markers include those that conferresistance to toxic chemicals (e.g., ampicillin resistance, kanamycinresistance, glyphosate resistance), complement a nutritional deficiency(e.g., uracil, histidine, leucine), or impart a visually distinguishingcharacteristic (e.g., color changes or fluorescence).

“Transcription” means the process of producing an RNA sequence copy froma DNA sequence template.

“Transformation” means a process of introducing an exogenous nucleicacid sequence (e.g., a vector, recombinant nucleic acid molecule) into acell or protoplast in which that exogenous sequence is incorporated intoa chromosome or is capable of autonomous replication.

“Transgenic plant” means a plant into which exogenous nucleic acidsequences are integrated.

“Vector” means any agent such as a plasmid, cosmid, virus, autonomouslyreplicating sequence, phage, or linear or circular single-stranded ordouble-stranded DNA or RNA nucleotide sequence, derived from any source,capable of genomic integration or autonomous replication, comprising anucleic acid molecule in which one or more nucleic acid sequences havebeen linked in a functionally operative manner. Such recombinant nucleicacid constructs or vectors are capable of introducing a 5′ regulatorysequence or promoter region and a selected DNA sequence into a cell insuch a manner that the DNA sequence may be transcribed into a functionalmRNA, which may subsequently be translated into a polypeptide orprotein. Recombinant nucleic acid constructs or recombinant vectors maybe constructed to be capable of expressing antisense RNAs, in order toinhibit translation of a specific RNA of interest.

Methods to Reduce Expression

Reduced expression of an endogenous gene in plants is achievable by avariety of means, including expression of an antisense sequence asdisclosed in U.S. Pat. No. 5,107,065, incorporated in its entirety,herein by reference. An antisense sequence is derived from the complete(full length) coding sequence of the gene or a fragment thereof. Anantisense sequence may also be a nontranslated portion of an endogenousplant gene, such as an intron, a 5′ nontranslated leader region or a 3′untranslated terminator or polyadenylation region of the gene as itexists in plants. Expression of a transgenic antisense sequence allowsfor the down-regulation of the specific endogenous plant gene. Antisenseregulation involves an antisense RNA sequence introduced into the cell,preferably under control of a strong promoter. The plant expressionvector contains the appropriate leader, termination, and processingsequences for expression of an RNA transcript in transgenic plants. Thetransgene antisense RNA sequence interacts with the endogenous sensemRNA to affect the transcription, processing, transport, turnover,and/or translation of the endogenous sense mRNA. Antisense inhibitionwas first reported in electroporation of carrot protoplasts withantisense and sense constructs containing the CAT reporter gene resultedin varying inhibition of CAT activity dependent on promoter strength(Ecker et al., Proc. Natl. Acad. Sci. U.S.A. 83: 5372–5376, 1986). Astable inheritable antisense effect was first reported in tobacco usingthe NOS transgene (Rothstein et al., Proc. Natl. Acad. Sci. U.S.A. 84:8439–8943, 1987). Constitutive expression of antisense chalcone synthase(CHS) in transgenic tobacco and petunia plants decreased endogenous CHSRNA and protein activity demonstrating the application of thistechnology in regulating endogenous gene expression (van der Krol etal., Nature 333: 866–869, 1988; van der Krol et al., Plant MolecularBiology 14: 457–466, 1990). The technology is extended to show seedspecific modulation of gene expression (versus leaf-specific modulation)using the B-conglycinin promoter to drive antisense expression of GUSmRNAs in transgenic tobacco (Fujiwara et al., Plant Mol. Biol. 20:1059–1069, 1992). The potential commercial value of antisense technologywas first realized when transgenic tomato plants expressing antisensepolygalacturonase (PG, an enzyme which partially solublizes cell wallpectin) showed a delay in fruit ripening (Smith et al., Nature 334:724–726, 1988). Antisense technology has since been used to alter theexpression of many plant genes, including ribulose bisphosphatecarboxylase oxygenase in tobacco (Rodermel et al., Cell 55: 673–681,1988), granule-bound starch synthase in potato (Visser et al., Mol. Gen.Genet. 225: 289–296, 1991), a photosystem II polypeptide in potato(Stockhaus et al., EMBO J. 9: 3013–3021, 1990), and TOM5 in tomato (Birdet al., Biotechnol. 9: 635–639, 1991), and PPO as described above.

Antisense sequence expression in plants has also been useful to alterplant development via the regulation of plant hormone biosyntheticpathways and relative hormone levels. For example, expression ofantisense ACC synthase and ACC oxidase RNA have been shown to inhibitfruit ripening in transgenic tomato (Oeller et al., Science 254:437–439, 1991; Hamilton et al., Nature 346: 284–287, 1990), andcantaloupe (Ayub et al., Nature Biotechnol. 14: 862–866, 1996).Expression of an antisense 7 transmembrane domain (7TM) receptorhomologue (GCR1) RNA reduced sensitivity to cytokinins in roots andshoots of transgenic Arabidopsis (Plakidou-Dymock et al., Current Biol.8: 315–324, 1998). Expression of antisense prosystemin severelydepressed systemic wound inducibility proteinase inhibitor synthesis intransgenic tomato and decreased resistance against insects (Schaller etal., Bioessays 18: 27–33, 1996). Expression of antisense catalase RNAsaccumulated high levels of PR-1 proteins and showed enhanced resistanceto tobacco mosaic virus (Takahashi et al., Plant J. 11: 993–1005,1997)in transgenic tobacco. Thus, much success has been achieved usingantisense technology to regulate biosynthetic pathways and hormonelevels in plants. In this way, reduction in endogenous PPO levels isinduced by constitutive or by the tissue-specific antisense inhibitionof expression of the endogenous PPO mRNA molecule.

Another way of reducing PPO levels and thereby reducing diseasesusceptibility is through homology-dependent gene silencing(cosuppression) of PPO genes. Specifically, overexpression of PPO mRNAcan be used to decrease PPO levels. Cosuppression, also known as cosensesuppression, homology-dependent gene silencing, repeat-induced genesilencing, et cetera, is the inactivation of a gene in a cell where itis normally functional (for reviews see Baulcombe et al., CurrentOpinion Biotechnol. 7: 173–180, 1996; Meyer et al., Annu. Rev. PlantPhysiol. Plant Mol. Biol. 47: 23–48, 1996; Matzke et al., Plant Physiol.107: 679–685, 1995). A description of cosuppression in plants may befound in U.S. Patents 5,034,323, 5,231,020, and 5,283,184, allincorporated in their entirety herein by reference. Transgene inducedcosuppression in plants has been shown to have useful effects whichinclude reduced impact of viral infection, fruit ripening, affectingflower color, inactivation of infecting transposons andretrotransposons, and editing aberrant RNA transcripts (Smyth et al.,Current Biol. 7: 793–795, 1997; Napoli et al., Plant Cell 2: 279–289,1990). Many examples of cosuppression have been reported in theliterature: sense suppression of caffeic acid O-methyltransferaseresulted in altered stem coloration of aspen (Tsai et al., PlantPhysiology 117: 101–112, 1998); cosuppression of a lipoxygenase isozyme(LOX2) resulted in transgenic Arabidopsis plants unable to accumulatejasmonic acid following wounding (Bell et al., Proc. Natl. Acad. Sci.U.S.A. 92: 8675–8679, 1995); cosuppression of phytochrome-regulatedchlorophyll α/β 140 RNA levels in Arabidopsis (Brussian et al., PlantCell 5: 667–677, 1993); cosuppression of a pea cDNA encodinglight-activated chloroplast NADP-malate dehydrogenase in transgenictobacco (Faske et al., Plant Physiol. 115: 705–715, 1997); cosuppressionof Flaveria bidentis NADP-MDH via heterologous sorghum NADP-MDH cDNAdespite only about 71% sequence homology (Trevanion et al., PlantPhysiol. 113: 1153–1163, 1997); cosuppression of a proline-richglycoprotein (TTS) involved in pollen tube growth in transgenic tobacco(Cheung et al., Cell 82: 383–393, 1995); cosuppression of phenylalanineammonia-lyase (PAL) in transgenic tobacco (Elkind et al., Proc. Natl.Acad. Sci. U.S.A. 87: 9057–9061); and cosuppression of two MADS boxfloral binding protein genes (FBP7 and FBP11) in petunia (Colombo etal., Plant Cell 9: 703–715, 1997). Cosuppression of a gene or sequencefor PPO expression will provide the same result as antisense regulationof these same PPO sequences or genes.

The present invention provides methods for reducing PPO expression andantisense oligonucleotides and polynucleotides complementary to any DNAsequence encoding PPO in potato plants. Such antisense oligonucleotidesshould be at least about six nucleotides in length to provide minimalspecificity of hybridization, and may be complementary to one strand ofDNA or to mRNA encoding PPO (or to a portion thereof), or to flankingsequences in genomic DNA which are involved in regulating PPOexpression. The antisense polynucleotides may be as large as manynucleotides, and may extend in length up to and beyond the full codingsequence for which it is antisense. The oligonucleotides andpolynucleotides can either be DNA or RNA. These antisense nucleotidesmay also be of chimerical source, single- or double-stranded. Thesenucleotides may also be prepared artificially by chemical synthesis. Theaction of the antisense nucleotide may result in specific alterationand/or primarily inhibition, of PPO gene expression in potato cells, asdiscussed above. Although one method of successful alteration of PPOgene expression will be achieved by introducing a full length cDNA clonegene in an antisense orientation, introduction of partial sequencestargeted to specific regions of the sequences can be effective as well.

With antisense and co suppression methods, reduced expression levels maybe achieved but with a low efficiency in transformation events. Nearcomplete target gene suppression may occur in as few as five to tenpercent of the transgenic events. Experience has shown it to occur forthe PPO gene using antisense technology in about five percent of theevents. A more recent development in gene suppression is a new methodwhich results in a higher efficiency of transformation, that is, highernumbers of events with high levels of suppression from each course oftransformation. This method uses a transgene which is composed ofinverted repeat sequences derived from the target gene, intended tocreate double-stranded mRNA via mRNA hybridization due toself-complementarity. This double-stranded RNA method (or “dsRNA”) hasbeen previously reported for genes other than PPO (Waterhouse et at.,Proc. Natl. Sci. USA, 95:13959–13964, 1998; Waterhouse et al., PlantMol. Biol., 43:67–82, 2000). The inverted repeat sequences may representa full or partial coding region, or any combination thereof, of thetarget gene, and can be fused either at 5′ or 3′ ends forming ahead-to-head or tail-to-tail type of structure. Transgenic plantexpression of the resultant inverted repeat gene fusion is under thecontrol of a single promoter and a single transcriptional terminator,and is thereby intended to create double-stranded messenger RNA (mRNA).

For this inverted repeat approach, the example below describes in moredetail the use of this efficient method of gene inactivation for PPO, inwhich a transgene is composed of inverted repeat sequences of the targetgene (potato PPO). Transgenic plant expression of the resultant invertedrepeat gene fusion is under the control of a single tuber-specificpromoter and a single transcriptional terminator. This inverted repeatdesign is intended to create double-stranded tuber PPO mRNA. Theconstruct design using inverted repeat sequences of the potato tuber PPOgene nearly completely inactivates potato tuber PPO gene expression(85–100% reduction in tuber PPO activity) in 87% of all transgenicevents evaluated for the potato cultivar Ranger Russet. This invertedrepeat transgene design is far superior to antisense technology in termsof the degree of PPO gene inactivation, as well as percentage of eventsin which the tuber PPO gene expression is highly inactivated.

Plant Transformation and Regeneration

Plants may be transformed by any of a variety of methods known to thoseskilled in the art such as by Agrobacterium transfection or biolisticsmethods. Potatoes are preferably transformed by the use of Agrobacteriumtransfection. A plasmid useful in any transformation method willpreferably contain a selectable marker to aid in elimination ofnontransformed plant cells. Plants transformed with one of thedown-regulating sequences described above may be assayed for PPOexpression levels. Levels of PPO protein can be measured using a PPOenzymatic activity assay. PPO levels which are no more than 20% of thenatural level will perform best in the present invention. Morepreferably, such levels will be no more than 15% of the natural level.Even more preferably such levels will be no more than 5% of the naturallevel. The plants which are amenable to transformation and use in thepresent invention are many. Examples include, but are not limited to,potato, sweet potato, banana, apple, avocado, broccoli, cauliflower,lettuce, grapevine, tobacco, bean, peach, pear and apricot. Each ofthese plants may be transformed by one of ordinary skill in the artusing known methods.

Regeneration will be conducted in obtaining a whole plant from thetransformation process. The term “regeneration”, means growing a wholeplant from: a plant cell, a group of plant cells, or a piece of tissue.Plant parts obtained from the regenerated plant in which expression of aPPO gene has been altered, such as leaves, flowers, seeds, fruit and thelike are within the definition of “plant” as stated above, and areincluded within the scope of the invention. Progeny and variants andmutants of the regenerated plants are also included, especially if theseparts comprise the introduced DNA sequences.

Disease Control

What is described here is a new method for disease control that is basedon the modification of parts of the phenylpropanoid pathway. Thispathway, initiated by the deamination of phenylalanine, leads to thebiosynthesis of multiple lignins, flavonoids, coumarins, benzoic acidsand esters such as chlorogenic acid.

Importantly, decreased levels of chlorogenic acid were previously shownto be associated with increased susceptibility to, e.g., P. infestans inpotato (Kening et al., 1995), Cercospora nicotianae in tobacco (Maher etal., Proc. Natl. Acad. Sci., USA 91: 7802–7806, 1994), and Moniliniafructicola in peach (Wang et al., Phytopathology 87: S101, 1997; Bostocket al., Phytopathology 87: S101, 1997). Chlorogenic acid or otherphenylpropanoid products may also provide either a direct antifungalactivity or limit pathogen-induced oxidative stress through theirantioxidant, iron-chelating or protease inhibitor-binding activities(Yamasaki and Grace, FEBS Lett., 1998, Feb. 6, 422(3): 377–380, 1998;Yoshino and Murakami, Anal Biochem. March 1, 257 (1): 4044, 1998; Feltonet al., J. Insect Physiol. 35: 981–990, 1989).

The in vivo concentrations of free phenolics such as chlorogenic acidare, in part, dependent on the activity of PPO which catalyzes theoxidation of chlorogenic acid and other phenolics to quinones uponwound-induced release from chloroplasts. It has been proposed that lowPPO levels may trigger expression of phenylalanine ammonia lyase (PAL),the rate-limiting enzyme in the biosynthesis of phenolics such aschlorogenic acid (Mayer et al., Annu. Rev. Plant Physiol. Plant Mol.Biol. 47: 23–48, 1996; Smith and Rubery, Planta 151, 535–540, 1981).

As compared to the PPO levels found in above-ground parts of plants,there is no apparent role for the up to 30-fold higher PPO levels inpotato tubers (Thygesen et al., Plant Physiol., 109: 525–531, 1995). Infact, these high concentrations of PPO lead to a rapid enzymaticbrowning upon wounding which can greatly reduce the agronomic value ofpotato tubers.

The present invention surprisingly demonstrates that plants can be madeto demonstrate resistance to the highly virulent US-8 genotype of P.infestans through genetic engineering. One possible mode of thisaccomplishment may be that decreased concentrations of PPO result in anincreased accumulation of free phenolics such as chlorogenic acid and,subsequently, trigger enhanced disease resistance against a variety offungal pathogens.

Promoters

In order for a newly inserted gene or DNA sequence, in the case ofantisense DNA, to be transcribed, resulting in an antisense RNAmolecule, preferably proper regulatory signals should be present in theproper location with respect to the coding or antisense sequence. Theseregulatory signals may include a promoter region, a 5′ non-translatedleader sequence and a 3′ polyadenylation sequence as well as enhancersand other known regulatory sequences. The promoter is a DNA sequencethat directs the cellular machinery to transcribe the DNA to produceRNA. Promoters useful in the present invention include those that conferappropriate cellular and temporal specificity of expression. Suchpromoters include those that are constitutive or inducible,environmentally- or developmentally-regulated, or organelle-, cell-, ortissue-specific.

Promoters which are useful in the present invention are those which willinitiate transcription in tissues in which polyphenol oxidase isproduced. Examples of such tissues include tuber tissues, fruit tissues,seed tissues, root tissues, flower tissues, and leaf tissues. Examplesof promoters useful in the present invention include, but are notlimited to promoters for granule bound starch synthases, soluble starchsynthases, ADP glucose pyrophosphorylases, sucrose synthases, starchbranching enzymes, starch debranching enzymes, polyphenol oxidases,sporamin proteins, and patatin proteins (Class I). Examples oftuber-specific promoters useful in the present invention include thegranule-bound starch synthase (GBSS) promoter from potato, the SporaminApromoter from sweet potato (Ohta et al., Mol Gen Genet. 225:369–378,1991), the TFM7 tomato fruit-specific promoter (Santino et al., PlantMol Biol, 33:405–416, 1997), and the ADP-glucose pyrophosphorylase smallsubunit (smADPGPP) promoter from potato (Nakata and Okita, Mol GenGenet, 250:581–592, 1996). Examples of constitutive promoters useful inthe present invention include the e35S promoter from the CauliflowerMosaic Virus (CaMV promoter) (Odell et al. (1985) Nature 313: 810), andthe ³⁵S promoter from the Figwort Mosaic Virus (FMV promoter) (Richinset al. (1987) NAR 20: 8451). These two are distantly relatedcaulimovirus promoters and are among the strongest promoters commonlyused in plant transformation.

Polyadenylation Signal

The 3, non-translated region of the chimeric plant gene contains apolyadenylation signal which functions in plants to cause the additionof polyadenylate nucleotides to the 3′ end of the RNA. Examples ofsuitable 3′ regions are the 3′ transcribed, non-translated regionscontaining the polyadenylated signal of Agrobacterium the tumor-inducing(Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and plantgenes like the 3′ non-translated region of the pea rbcS-E9 gene.

Polyphenol Oxidase DNA Sequence Sources

The PPO sequence used in the DNA constructs for plant transformation inthe present invention may be any PPO DNA sequence. It is not limited tothe potato tuber PPO sequence for the isoform product, although it ispreferred for potato tuber-specific inhibition of PPO. Genomic or cDNAPPO sequences from other plant sources and/or from other non-tubertissues may be moved into plasmids containing plant-appropriateregulatory sequences and used in the present invention. Any PPO sequencethat comprises any portion of its open reading frame, or any portion ofits 5′ or 3′ untranslated region, or any combination of portions or theentirety of its open reading flame plus portions or the entirety of itsuntranslated regions, may be moved into plasmids containingplant-appropriate regulatory sequences and used in the presentinvention.

A PPO cDNA sequence (PPO-P1) for a potato leaf isoform polyphenoloxidase has been previously identified in which the gene source was theKatahdin cultivar (Hunt, M. D., et al., 1993, Plant Mol. Biol.21:59–68). Additionally, the gene for the tuber predominant plustuber-specific PPO isoform was cloned from the Ranger Russet potatocultivar, based upon expression analysis of the various potato PPOisoforms, specifically of the major tuber isoform POT32 (Thygesen, P.W., et al., 1995, Plant Physiol. 109:525–531). Using the GenBanksequence (accession U22921) for POT32, specific primers were made andthe gene was amplified by PCR from Ranger Russet genomic DNA. Sequencingof the entire open-reading-frame of the PCR gene product revealed thatthe sequence from the Ranger Russet cultivar is 98% identical to thepublished POT32 gene which is from the Norchip cultivar. Thus, theputative tuber-predominant, putative tuber-specific Ranger Russet PPO,referred to hereafter as the “Ranger Russet tuber PPO sequence” or“RR-PPO sequence,” was chosen to be used in the examples below.

Transgenic potato tubers containing less than 20% of wild-type PPOlevels display enhanced resistance against P. infestans. It will berecognized by one skilled in the art that these tubers will also displayenhanced disease resistance against certain other fungal pathogens thatinfect potato tubers. Furthermore, the experiments detailed belowsuggest that suppression of PPO may result in the control of fungalpathogens of potatoes including, but not limited to, Spongospora(powdery scab), Rhizoctonia (black scurf), Fusarium (dry rot),Verticillium (Verticillium wilt), Alternaria (early blight),Polyscytalum (skin spot), Sclerotium (white mold), Rosellinia (blackrot), Helicobasidium (violet root rot), Macrophomina (charcoal rot), andHelminthosporium (silver scurf). The methods may also result in controlof fungal pathogens including, but not limited to, Phytophthora,Cercospora and Monilinia, on other plants.

After a general description of the present invention, the followingspecific examples are presented to further depict the same in a specificmanner. These examples are provided by way of illustration, and are notin any way intended to limit the present invention. Therefore, theseexamples can not be construed as to limit the scope of the invention.

EXAMPLES Example 1 Construction of Antisense Plant Vectors

In order to obtain tuber-specific expression of the potato leaf cDNA, aplant expression vector, pMON21624, which contained the PPO-P1 sequencewas constructed as follows: The PPO-P1 sequence was isolated from apotato leaf cDNA library as a SacI-BglII fragment, and fused inantisense orientation to the 3′ end of the e35S promoter and 5′ end ofthe E9 3′ nontranslated polyadenylation region by ligation into aSacI-BglII sites of a double-bordered binary Ti plasmid vector. Thevector contained the e35S/NPTII/NOS 3′ selectable marker cassette, whichconfers plant resistance to kanamycin. From the resultant plasmid,pMON21621, the e35S promoter was removed as a HinDIII-BglII fragment andreplaced with the potato GBSS promoter via ligation as a HinDIII-BglIIfragment to produce pMON21622, which contains the GBSS/antisensePPO-P1/E9, 3′ cassette. The e35S/NPTII/NOS 3′ cassette was then excisedas a NotI-XhoI fragment from pMON21622. The FMV/CTP2-CP4/E9 3′selectable marker cassette which confers plant tolerance to glyphosate(Padgette et al., Herbicide Res. Crop: Agricultural, Environmental,Economic, Regulatory and Technical Aspects, CRC Press, 53–84, 1996), wasisolated as a NotI-SalI fragment from pMON17314, which is a pUC-basedEscherichia coli cloning vector, and ligated into the NotI-XhoI sites ofpMON21622 to produce the final plasmid, pMON21624.

The RR-PPO sequence, which was amplified by PCR from Ranger Russetgenomic DNA, was cloned into the HindIII site of pMON38201, which is apBluescriptII-SK(−) (Stratagene)-based cloning vector with a modifiedmulticloning polylinker. The resultant vector, pMON21646, served as theRR-PPO sequence source for all subsequent plant vector constructions. Inorder to obtain tuber-specific expression of the tuber PPO sequence,three different plant expression vectors which contained the RR-PPOsequence were constructed.

The first expression vector, pMON21652, was built as follows: The RR-PPOsequence was isolated as a SalI-BamHI fragment from pMON21646, while theE9 3′ nontranslated polyadenylation fragment was isolated from pMON21647(not described herein) as an XhoI-NotI fragment. pMON17269 is adouble-bordered binary Ti plasmid vector which contains the smADPGPPpromoter, plus the e35S/NPTII/NOS 3′ selectable marker cassette. Inorder to fuse the smADPGPP promoter to the RR-PPO sequence in antisenseorientation, pMON17269 was digested with BglII and NotI. In a tripleligation, the BamHI site at the 3′ end of the RR-PPO sequence wasligated to the BglII site at the 3′ end of the smADPGPP promoter, theSalI site of the RR-PPO sequence was ligated to the XhoI site at the 5′end of the E9 3′ fragment, and the NotI site of the E9 3′ fragment wasligated with the NotI site of pMON17269. The resultant plasmid,pMON21650, containing the smADPGPP/antisense RR-PPO/E9 3′ cassette, wasthen digested with NotI-XhoI in order to excise the e35S/NPTII/NOS 3′cassette. The FMV/CTP2-CP4/E9 3′ selectable marker cassette was isolatedas a NotI-SalI fragment from pMON17314, which is a pUC-based E. colicloning vector, and ligated into the NotI-XhoI sites of pMON21650 togive the plasmid, pMON21652.

The second expression vector, pMON21656, was constructed as follows:pMON31813 is a cloning vector which contains the SpoApromoter/polylinker/NOS3′ cassette flanked by NotI sites. The RR-PPOsequence was isolated from pMON21646 as a SacI-BamHI fragment andligated in antisense orientation into the BglII-SacI sites of pMON31813producing pMON21655. pMON34018 is a double-bordered binary Ti plasmidvector which contains the FMV/CTP2-CP4/E9 3′ selectable marker cassette.The SpoA/antisense RR-PPO/NOS 3′ cassette was excised from pMON21655 asa NotI fragment, and ligated into the unique, dephosphorylated NotI siteof pMON34018, to produce the plasmid, pMON21656.

The third expression vector, pMON38914, was constructed as follows: TheRR-PPO sequence was isolated from pMON21646 as a KpnI-BamHI fragment,while the TFM7 promoter was isolated from tomato library as aHinDIII-BglII fragment. pMON10097, which is a double-bordered binary Tiplasmid vector which contains the E9 3′ nontranslated polyadenylationfragment between the borders, was digested with KpnI and HinDIII. In atriple ligation, the BamHI site at the 3′ end of the RR-PPO gene wasligated to the BglII site at the 3′ end of the TFM7 promoter, the KpnIsite of the RR-PPO gene was ligated to the KpnI site at the 5′ end ofthe E9 3′ fragment, and the HinDIII site of the TFM7 promoter fragmentwas ligated with the HinDIII site of pMON10097. The resultant plasmid,pMON21651, contained the TFM7/antisense RR-PPO/E9 3′ cassette and wasdigested with NotI-XhoI. The FMV/CTP2-CP4/E9 3′ selectable markercassette was isolated as a NotI-SalI fragment from pMON17314, which is apUC-based E. coli cloning vector, and ligated into the NotI-XhoI sitesof pMON21651 to produce the plasmid, pMON38914.

Example 2 Transformation, Expression and Regeneration of Potato

The constructs pMON21624, pMON21652, pMON21656, and pMON38914 wereindependently mobilized into disarmed ABI Agrobacterium tumefaciensstrain by electroporation using a Bethesda Research LaboratoriesCell-Porator according to the manufacturer's recommended protocol.Electroporated cells were allowed to recover in LB broth with shaking(200 rpm) at 30° C. for 2 hours. Transformed A. tumefaciens cells wereselected by plating out the electroporated cells on LB agar containing25 ug/ml chloramphenicol, 50 ug/ml kanamycin, and 75 ug/mlspectinomycin.

Russet Burbank and Ranger Russet potato cultivars underwentAgrobacterium-mediated transformation using a glyphosate selectiontransformation protocol as described in U.S. Pat. No. 4,970,168,incorporated in its entirety herein by reference. Russet Burbankcultivar was transformed with only pMON21624, while Ranger Russetcultivar was independently transformed with pMON21652, pMON21656, andpMON38914.

Specifically, sterile shoot cultures of each of the cultivars weremaintained in vials containing 10 ml of PM medium (Murashige and Skoog(MS) inorganic salts, 30 g/l sucrose, 0.17 g/l Na H.sub.2PO.sub.4H.sub.2 O, 0.4 mg/l thiamine-HCl, and 100 mg/l myo-inositol,solidified with 2 g/l Gelrite at pH 6.0). When shoots reachedapproximately 5 cm in length, stem internode segments of 7–10 mm wereexcised and inoculated for 15 min in a square petri dish, with anAgrobacterium tumefaciens overnight liquid culture diluted to an opticaldensity of 0.2–0.33. The stem explants were co-cultured for three daysat 23° C. on a sterile filter paper placed over 1.5 ml of a tobacco cellfeeder layer overlaid on 1/10 P medium ( 1/10 strength MS inorganicsalts and organic addenda without casein as in Jarret, et al. (1980), 30g/l sucrose and 8.0 g/l agar). About 50 explants were placed perco-culture plate. Following co-culture, the explants were transferred tofull strength P-1 medium for callus induction, composed of MS inorganicsalts, organic additions as in Jarret, et al. (1980) with the exceptionof casein, 10 mg/l AgNO₃, 5.0 mg/l Zeatin Riboside, and 0.1 mg/lnaphthaleneacetic acid (NAA) (Jarret, et al., 1980), for 2 days.Carbenicillin (500 mg/l) is included to inhibit bacterial growth.Explants were subsequently transferred onto callus induction media whichcontained glyphosate (0.025 mM) to select for transformed cells. Afterfour weeks the explants were transferred to shoot induction media of thesame composition but with 0.3 mg/l gibberellic acid (GA3) replacing NAA(Jarret, et al., 1981) to promote shoot formation. Explants weretransferred to fresh shoot induction media every 4 weeks for 12 weeks.Shoots begin to develop approximately two weeks after transfer to shootinduction medium; these were excised and transferred to vials of Pmedium for rooting every 4 weeks for 12 weeks.

Example 3 Oxidative Browning Assay and Catechol Assay for PPO Activityin Potato Tubers

Transgenic Russet Burbank and Ranger Russet lines were initiallyscreened for tuber PPO activity using greenhouse-grown mini-tubers;Transgenic plantlets with sufficient root development in tissue culturewere transplanted to soil which consisted of 2 parts Metro-350, 1 partfine sand, 1 part Ready-Earth in 12 inch wide pots. The plantlets weregrown in a greenhouse in which conditions throughout a 4–5 month growthperiod included a 15 hr photoperiod, 40–60% relative humidity,fertilization with Peter's Special 20–20–20, and 24-27° C. day/13–16° C.night incubation. At 2.5 months, fertilization was stopped, and uponsenescence, tubers were harvested and stored at 4° C. until evaluation.

Mature greenhouse mini-tubers, as well as field-grown tubers weremeasured for PPO activity first by an Oxidative Browning Assay and thenby a Catechol Assay. The Oxidative Browning Assay of tuber homogenatesrelies only upon endogenous tuber phenolic compounds as substrates forPPO. With this assay, no additional substrate is added to the tuberhomogenates, and the natural browning color is allowed to develop over20 hours. The Catechol Assay of tuber homogenates involves addition ofcatechol (DOPA substrate analog) to the extract, followed by monitoringthe rapid increase in color development spectrophotometrically. Themajority of lines with wild type tuber PPO activity levels wereeliminated by the Oxidative Browning Assay; therefore, the CatecholAssay was performed as a more stringent confirmation assay on transgeniclines initially identified as having low PPO activity by the OxidativeBrowning Assay.

1. PPO-Oxidative Browning Assay Of Tuber Homogenates. For eachtransgenic line, measurements were performed on samples of tuber tissue(approximately 1–5 grams) extracted by a #8 cork punch from the centerof each tuber. Tuber tissue was homogenized using a Polytron (KinematicaPolytron, Taiwan) on speed five for approximately 30 seconds, in 20 mMsodium acetate buffer, pH 5.2, at a 1:5 (w:v) tissue to buffer ratio.Proteins (usually 5 μL) were analyzed in duplicate by Bradford assay.The homogenate was allowed to oxidize at 22° C. for 20 hours in the sameround bottom tube, centrifuged at 12,000 rpm for 5 minutes, and decantedfor optical density measurements. Optical density was directly measuredat 475 nm. Units of oxidative browning rate were calculated as opticaldensity after 20 hours divided by mg of protein on a per ml basis(divided by mg protein/mL). In the case of greenhouse mini-tubers,tyrosine was optionally added to the homogenates immediately afterhomogenization at a final concentration of 2.0 mM in order to intensifythe brown color development after 20 hours.

2. PPO-Catechol Assay of Tuber Homogenates. For each transgenic line,measurements were performed on samples of tuber tissue (approximately1–5 grams) extracted by a #8 cork punch from the center of each tuber.Tuber tissue was homogenized using a Polytron on speed five forapproximately 30 seconds, in 20 mM sodium acetate buffer, pH 5.2, at a1:5 (w:v) tissue to buffer ratio, and centrifuged at 12,000 rpm for 3min. Extracted samples were desalted in duplicate (175 μL each) usingBoehringer Mannhein Protein Quick-Spin desalting columns equilibratedwith 20 mM sodium acetate buffer, and assayed in duplicate for PPOactivity using 10 mM final concentration catechol as substrate. Proteinwas measured in duplicate on 10 μL the desalted samples by Bradfordassay. One ml of the PPO reaction mixture contained 100 μL of 100 mMcatechol, 100 μg of total extracted protein (usually 50–100 μL ofdesalted extract), and a volume of 20 mM sodium acetate buffer, pH 5.2(usually 800–850 μL) to bring total reaction volume to 1.0 mL. Themixture was rapidly mixed and the change in optical density wasmonitored at 420 nm for the first 2 minutes. PPO specific activity wascalculated as the change in OD at 420 nm/min/mg protein assayed.

Example 4 Bruise Barrel Assay for Black Spot Bruise Resistance

Transgenic Ranger Russet lines which demonstrated greater than a 50%reduction in tuber PPO activity via Catechol Assay of greenhousemini-tubers, were evaluated for black spot bruise resistance in fieldgrown tubers. Anti-black spot bruise field trials were conducted twicein two consecutive years at Parma, Id., Notus, Id., Hancock, Wis., andHermiston, Oreg. The trials consisted of either tissue culturepropagated plantlets, or first generation certified tuber seed. Alltrials consisted of 3 to 6 repetitions of 12 to 20 plants/seed piecesper repetition per transgenic line, with a complete randomized blockdesign. Tubers in the 6 to 12 ounce category were harvested at fullmaturity from all repetitions, and subsequently underwent a BruiseBarrel Assay for black spot bruise susceptibility. The Bruise BarrelAssay mimics commercial conditions of physical impact encountered duringharvest, shipping, and storage of potato tubers, thereby allowingidentification of transgenic lines with resistance to black spot bruise.

Bruise Barrel Assay. Field collected tubers were handled with care toprevent bruising, and were allowed to warm to 22° C. for 24 hours.Fifteen tubers per field repetition were placed in a motor-driven bruisebarrel apparatus, and the barrel was turned on and allowed to rotate forexactly 15 revolutions. The barrel was equipped with a counter set for15 revolutions in order to always stop the barrel exactly in the sameposition. Tubers were removed from the barrel and placed in a bucket,with each bucket representing a single field repetition consisting of 15tubers. The tubers were held at 22° C. for 7 days to allow black spotbruises to develop, at which point a Hobart peeler was employed topartially peel each repetition of 15 tubers. This process was referredto as an Abrasive Peel in which the Hobart peeler was run for exactly 30seconds with running water supplied. Tubers were removed and hand-peeledto complete the peeling process. All black spot bruises were immediatelycounted per tuber, taking care to not include shatter bruises, in orderto obtain a total count per 15 tubers. For each repetition, all 15peeled tubers were returned to the same bucket and held at 22° C. for 24hours. Tubers were removed from the buckets and individually assigned anAbrasive Peel Rating, based on the propensity of the entire tuber tissueto darken or discolor. Abrasive Peel ratings were based on the followingvisual color scale: 1=white or light yellow; 2=small areas of lightgray; 3=more or larger areas of light gray; 4=large areas of intensegraying, darkening, or blackening; 5=tuber blackened completely.

Three different variations of the Bruise Barrel Assay were employed.Bruise Barrel Assay 1 (BBA1) involved bruising groups of 15 tubers(usually three or more repetitions of 15 tubers per line) immediatelyafter harvest. The tubers were then allowed to sit at 22° C. for 7 days,followed by peeling and counting of black spot bruises. Bruise BarrelAssay 2 (BBA2) involved bruising the same number and repetitions oftubers immediately after harvest. However, the tubers were stored at 4°C. for 4 months, after which they were removed from cold storage andpeeled to count black spot bruises. Bruise Barrel Assay 3 (BBA3)involved storing the same number and repetitions of tubers first for 4months at 4° C., after which the tubers were removed from cold storage,bruised, allowed to sit at 22° C. for 7 days, and then peeled to countblack spot bruises.

Example 5 Analysis of Russet Burbank Potato Line

The transformation of Russet Burbank with pMON21624 generated six lines,designated Marie and RBHS90 lines, which demonstrated a significantreduction in greenhouse mini-tuber PPO activity levels from that ofnontransgenic Russet Burbank control (Table 1). These six Russet Burbanklines were grown at a certified seed facility in Islands Falls, Me., inorder to obtain disease-free field tubers to test in late blightresistance studies, as well as to measure for field tuber PPO activitylevels.

For each of the six transgenic Russet Burbank lines plus Russet Burbankcontrol, six tubers in the 6–8 ounce category per line were obtainedfrom the same plots in Island Falls, Me., in which samples were takenfor tuber PPO activity assay. To prepare the late blight tuber inoculum,P. infestans isolate A2 US8 was grown for 10 days at 18° C. on petriplates containing rye agar-A. Sterile distilled water suspensions ofsporangia from 40 plates were pooled and placed in a single Erlenmeyerflask and incubated at 8° C. for 3 hours to induce zoospore formation.The concentration of zoospores was determined with a hemacytometer andthe suspension was diluted to approximately 2×10⁴ zoospores/ml. Thisdiluted suspension also contained approximately 1000 sporangia/ml. Allsix tubers for each line were placed in 7-Way trays, which providedoptimum conditions for washing, drying, inoculating, and incubation ofthe tubers. Each tray held 6 tubers, and the tubers were washed with tapwater. When dry, the tubers were each wounded with a circular Flour Pegsteel comb, 1 inch diameter, which contained approximately 60 steelneedle-like teeth. The comb was pricked once through the skin of thetuber at its longitudinal center, and the zoospore suspension was evenlysprayed onto the wounded tubers with an atomizer. Immediately afterinoculation, the trays were incubated in a mist chamber at 17° C., 100%relative humidity for 24 hours, and then at 20° C., 80–90% relativehumidity for 12 days. Following incubation, the tubers were carefullypeeled completely by hand and were rated for disease severity of thetuber tissue. The results in Table 1 indicate a direct correlationbetween the reduction in field tuber PPO activity levels, and areduction in the percentage of disease severity area as well asreduction in the spread of disease into the tubers, as compared to thecontrol.

Transgenic tubers of lines MARIE-65, HS90–22 and HS90–25, whichcontained>20% of wild-type PPO levels were equally susceptible to P.infestans as controls, with at least 54% and 71% of the tuber surfacedisplaying disease symptoms at the day 12- and day 20-time points,respectively. Importantly, tubers of transgenic lines MARIE-72, HS90-23and HS90-07, with PPO levels below 20% of the levels in untransformedplants, displayed a significantly enhanced level of disease resistance.Twelve days post-infection, disease symptoms were, on average, reducedwith 36% in transgenic tubers derived from these ‘low PPO’ plantscompared to untransformed control tubers. Eight days later, ‘low PPO’tubers still displayed 28% less disease symptoms than the potato linescontaining higher concentrations of PPO. To confirm that ‘low PPO’levels were correlated with reduced disease symptoms, the spread of P.infestans-induced disease symptoms in the tubers was measured. As shownin Table 1, the depth of affected tissue in both controls and transgeniclines containing at least 20% of wild-type PPO levels was between 4.3and 4.8 mm, whereas disease symptoms were limited to 2.3–3.3 mm intubers of transgenic lines with less than 20% PPO. In examining thephysical appearance of the disease symptoms, transgenic ‘low PPO’ tubersclearly displayed less disease-induced symptoms than the Russet Burbankwild-type controls.

TABLE 1 Percent disease severity in Russet Burbank tubers inoculatedwith Phytophthora infestans isolate A2 US8, and corresponding percentreduction in tuber PPO activity for lines transformed with pMON21624.Spread of % Late Blight % Red. % Red. Disease severity disease in tubersin PPO in PPO area on tubers (depth in mm) Line ID Greenhouse¹ Field² 12DAI 20 DAI 12 DAI 20 DAI RB 0 0 61 77 4.3 5.0 Control HS90-23 84 99 3956 2.3 3.8 Marie-72 94 89 44 52 3.3 3.3 HS90-07 87 86 38 58 3.3 4.5HS90-25 71 76 54 71 4.8 4.8 Marie-65 65 31 69 80 4.8 4.5 HS90-22 26 7 6778 4.5 8.0 LSD 6.9 13 0.9 3.6 ¹= average of two mini-tubers evaluatedper line. ²= average of 4 tubers evaluated per line. Tubers were fromsame source as those used for P. infestans isolate US8 inoculations.

Example 6 Analysis of Ranger Russet Potato Line

The transformation of Ranger Russet with pMON38914 generated six lines,designated Gemini, which demonstrated a significant reduction ingreenhouse mini-tuber PPO activity levels from that of wild type RangerRusset control (Tables 2, 3, & 4). These 6 Ranger Russet lines underwentfield trial evaluations in Notus, Id., and Hancock, Wis., in which theywere evaluated for black spot bruise resistance using the BBA1, andBBA2. The black spot bruise data shown in Tables 2 and 3 indicate theselines demonstrate a strong correlation in the reduction of greenhousemini-tuber PPO activity with a significant reduction or elimination ofblack spot bruises in both BBA1 and BBA2 at both field trial locations.Significant reductions in Abrasive Peel ratings at both trial sites alsoindicated a decreased propensity for the tuber tissue to form blackmelanin pigment in the transgenic lines, due to the reduced activity ofPPO in the tuber.

TABLE 2 Black spot bruise resistant Ranger Russet lines: Field Trials atHancock, WI, and Notus, ID. BBA1 - Tubers bruised and evaluated atharvest. All bruise and abrasive peel data represent the average of 3repetitions of 15 tubers per repetition. % Red. Hancock, WI Notus, ID inPPO Mean # Abrasive Mean # Abrasive Line Greenhouse¹ Bruises Peel Rat.Bruises Peel Rat. Ranger Control 0 62 4.6 28 3.9 Gemini-087 98  0*  2.6* 0*  1.7* Gemini-108 81  0*  2.7*  0*  1.6* Gemini-180 73  0*  2.5*  4* 1.9* Gemini-200 69  0*  2.4*  0*  1.5* Gemini-157 20 44 4.4 29 4.0Gemini-191 9 51 4.5 34 4.0 *= Statistically different from control. ¹=average of three mini-tubers evaluated per line.

TABLE 3 Black spot bruise resistant Ranger Russet lines: Field Trials atHancock, WI, and Notus, ID. BBA2 - Tubers bruised at harvest, thenstored 4 months at 4° C., and then peeled for evaluation. All bruise andabrasive peel data represent the average of 3 repetitions of 15 tubersper repetition. % Red. Hancock, WI Notus, ID in PPO Mean # Abrasive Mean# Abrasive Line Greenhouse¹ Bruises Peel Rat. Bruises Peel Rat. RangerControl 0 90 4.3 22 4.0 Gemini-087 98  0*  3.0*  0*  2.4* Gemini-108 81 0*  3.3*  0*  2.6* Gemini-180 73  0*  3.1*  1*  2.5* Gemini-200 69  0* 3.0*  1*  2.5* Gemini-157 20 86 4.3 23 4.4 Gemini-191 9 87 4.2 18 4.1*= Statistically different from control. ¹= average of three mini-tubersevaluated per line.

These same six Ranger Russet Gemini lines were grown at a certified seedfacility in Island Falls, Me., in order to obtain disease-free fieldtubers to test in late blight resistance studies, as well as to measurefor field tuber PPO activity levels. Tubers obtained from all six RangerRusset lines grown in there demonstrated that reduction of mature fieldtuber PPO activity correlated closely with that observed for the samelines grown in the greenhouse (Table 4). The combination of black spotbruise resistance, plus field-level reduction of tuber PPO activitylevel provided sample test material for evaluation of resistance to thelate blight pathogen P. infestans isolate US8.

To confirm that strong reductions in PPO levels not only lead to areduction of disease symptoms but also result in reduced fungal growth,a “tuber slice fungal penetration” study was designed. For each of thesix transgenic Ranger Russet lines plus wild type Ranger Russet control,six tubers in the 6–8 ounce category per line were obtained from thesame field plots in Island Falls, Me., in which samples were taken fortuber PPO activity assay. To prepare the late blight tuber inoculum, P.infestans isolate US8 was grown for 10 days at 18° C. on petri platescontaining Rye Agar-A until the agar surface was completely covered.Tubers were cut at the center point to obtain one thick (1 cm)cross-sectional slice per tuber. Each tuber slice was placedhorizontally onto a square agar plug (0.5 cm width) containing P.infestans sporangia, and incubated in a sealed petri dish (100%humidity) at 20° C. Five days post-inoculation, the top surfaces of thetuber slices were analyzed phenotypically for the presence of P.infestans that had grown through the slices and subsequently sporulated.Each slice was measured for the percentage of disease severity areaacross the top surface. The tissue disease severity ratings in Table 4indicate a direct correlation between the reduction in field tuber PPOactivity levels, and a reduction in the percentage of disease severityarea across the upper surface of the slice. Importantly, it was alsofound that P. infestans sporulated vigorously on slices obtained fromRusset Burbank control tubers, thereby producing significant biomass(mycelia and spores), but that tuber slices from all ‘low PPO’ Geminilines contained hardly any P. infestans biomass (mycelia and spores)(data not shown).

TABLE 4 Percent disease severity on top surface of Ranger Russet cross-sectional tuber slices inoculated from the underside surface with P.infestans isolate A2 US8, and corresponding percent reduction in tuberPPO activity, for lines transformed with pMON38914. % Reduction %Reduction % Disease severity in PPO in PPO area on tuber Line IDGreenhouse¹ Field² slices: 5 DAI³ Ranger Control 0 0 59 Gemini-087 98 9927 Gemini-108 81 90 45 Gemini-180 73 86 45 Gemini-200 69 79 41Gemini-157 20 49 30 Gemini-191 9 0 64 ¹= average of three mini-tubersevaluated per line. ²= average of 5 tubers evaluated per line. Tuberswere from same source as those used for Phytophthora infestans isolateUS8 inoculations. ³= average of 6 tuber slices (one slice per tuber; 6tubers total) evaluated per line.

Example 7 Evaluation of Ranger Russet Lines in a Late Blight Field Trial

Three of the six Gemini lines described in Example 6 were chosen for alate blight resistance field trial, based upon their significantgreenhouse and field-level reduction of tuber PPO activity level plustheir resistance to black spot bruise, as demonstrated in Tables 2–4 ofExample 6. The objective of the study was to determine if ‘low PPO’,black spot braise resistant Ranger Russet lines exhibit field levelresistance to P. infestans, the causal agent of late blight.

FG1 seed of transgenic Ranger Russet lines Gemini 087, Gemini 108,Gemini 200, and standard nontransgenic Ranger Russet was produced at thesame certified seed facility in Islands Falls, Me. as mentioned inExample 6. The seed was planted in early June in a randomized completeblock design at the Brown Horticultural Research Farm near Corvallis,Oreg. Individual plots consisted of single rows, 25 hills in length,replicated 4 times. Potato plants were not treated with fungicides, andfoliar infection by P. infestans developed by late August. Foliardisease levels were rated periodically through the remainder of theseason, until vine kill in mid September. At harvest on September28^(th), all tubers were lifted to the soil surface and evaluated forvisual symptoms of late blight infection. Healthy and blighted tuberswere weighed separately, and the percentage blighted tubers wasdetermined by dividing the fresh weight of blighted tubers by the totalweight of all tubers.

The results of the tuber evaluation are presented in Table 5. All threetransgenic Gemini lines exhibited significantly lower percentages oflate blight infected tubers than Ranger Russet control. The level oflate blight resistance was similar in all three Gemini lines. The fieldtrial confirms that black spot bruise resistant Ranger Russet lines,which have a reduction in tuber PPO level, have increased fieldresistance to tuber infection by P. infestans, the causal agent of lateblight. The field level resistance to late blight pathogen P. infestansby the transgenic ‘low PPO’, black spot bruise resistant tuberscorrelates with the resistance observed for the tuber inoculationstudies described in Examples 5 and 6.

TABLE 5 The affect of natural field infection by P. infestans onincidence of late blight tuber infection in transgenic Ranger Russetpotato lines, Corvallis, OR - 2000. Total tuber wt per Wt of infected %Late blight Line plot (lbs) tubers (lbs) infected tubers Ranger 26.7 4.517.0 Gemini 087 24.6 1.0 4.9 Gemini 108 26.9 0.8 3.2 Gemini 200 25.7 1.45.3 LSD (p = 0.05) NS 2.0 8.5

Transgenic potato tubers generally containing less than 25% of wild-typePPO levels display enhanced resistance against P. infestans. Furtherevidence in support of this comes from late blight disease tests ontubers from transgenic Ranger Russet lines which are transformed withthe smADPGPP promoter, or the SpoA promoter, driving antisenseexpression of the RR-PPO (constructs pMON21652 and pMON21656,respectively). Tuber PPO analyses of transgenic Ranger Russet events,transformed with pMON21562 or pMON21656 have identified populations oflines with tuber PPO activity levels reduced by 50–100% when compared tothe wild type control (data not shown). In these tubers, significantlyenhanced levels of late blight resistance were observed when PPO levelswere reduced by 50% or greater, which indicates that other promoterssuch as the smADPGPP and SpoA promoters may perform equally as well, andeven better than the GBSS and TFM7 promoters in terms of diseaseresistance.

Example 8 Use of dsRNA Method

For plant vector construction, a nearly full length RR-PPO deletion genewas created within the unique HindIII and NruI sites of the RR-PPOopen-reading-frame, in which the first six nucleotides at the 5′ end ofthe open-reading-frame were omitted, and the last 148 nucleotides at the3′ end were omitted. The resultant RR-PPO deletion sequence consisted ofa 5′-HindIII, 3′-NruI gene fragment composed 1638 nucleotides. ThisRR-PPO deletion sequence was fused to an exact copy of itself at its 3′end by ligation of the NruI sites of both fragments. The resultantinverted repeat RR-PPO deletion sequence, otherwise referred to as the“double-PPO gene” was cloned behind the promoter for the small subunitof the ADP-glucose pyrophosphorylase gene from potato, in adouble-bordered binary Agrobacterium vector, which contained the CP4EPSP synthase gene cassette for selection of transgenic plants on aglyphosate-containing medium. The resultant vector, pMON38293, nowcontained the double-PPO gene cassette as follows:smADPGPP/sensePPO-antisensePPO/E93′. See FIG. 5 for the plasmid map ofpMON38293.

Potato cultivar Ranger Russet was transformed with pMON38293 viaAgrobacterium—mediated transformation using glyphosate selectiontransformation, generally as described above. Ranger Russet pMON38293transgenic lines were assigned the field name “Peru”.

Two populations of Peru Ranger Russet lines transformed with pMON38293were evaluated for tuber PPO activity. The first group consisted of 30lines, while the second group consisted of 23 lines. The results of thePPO tyrosine and catechol assays, as well as the percent reduction inPPO activity from the Ranger Russet control based upon the specificactivity of the catechol assay, are presented in Table 6.

TABLE 6 Catechol % Reduc. Tyrosine Assay Activity Line # Assay Spec.Act. From Control Assay Request 55 Control 5 0.511 Control 5 0.510 105330 0.001 99.8 10534 0 0.003 99.4 10535 5 0.412 19.4 10536 0 0.005 99.010537 0 0.026 94.9 10538 0 0.015 97.1 10539 5 0.456 10.8 10540 0 0.00698.8 10541 0 0.021 95.9 10542 0 0.038 92.6 10543 0 0.025 95.1 10544 00.005 99.0 10546 0 0.004 99.2 10547 3 0.511 0.0 10548 0 0.034 93.3 105490 0.002 99.6 10550 0 0.041 92.0 10551 0 0.005 99.0 10552 3 0.365 28.610554 0 0.006 98.8 10555 0 0.003 99.4 10556 0 0.005 99.0 10557 0 0.00599.0 10558 0 0.004 99.2 10559 0 0.008 98.4 10560 0 0.013 97.5 10561 00.022 95.7 10562 0 0.021 95.9 10563 0 0.004 99.2 10564 0 0.003 99.4Assay Request 58 Control 5 0.336 Control 5 0.339 10565 0 0.002 99.410567 0 0.004 98.8 10568 0 0.010 97.0 10569 0 0.020 94.0 10570 0 0.01097.0 10572 0 0.020 94.0 10573 5 0.346 −3.0 10574 1 0.020 94.0 10575 40.238 29.2 10576 0 0.020 94.0 10577 0 0.007 97.9 10580 0 0.040 88.110581 0 0.020 94.0 10582 0 0.010 97.0 10584 0 0.050 85.1 10585 1 0.05085.1 10586 5 0.159 52.7 10587 0 0.010 97.0 10588 0 0.040 88.1 10589 00.040 88.1 10590 0 0.010 97.0 10591 0 0.040 88.1 10592 0 0.040 88.1

The results indicate that 46 out of 53 total independent transgenicevents (lines), or 87%, have 85% or better reduction in tuber PPOspecific activity (catechol assay), and that 39 out of 53 total lines,or 74%, have 90–100% reduction in tuber PPO specific activity (catecholassay), as compared to the wild type Ranger Russet control. Many linesdemonstrate nearly 100% reduction in tuber PPO specific activity. Theresults of the tyrosine assay also closely correlate with those of thecatechol assay in that lines which had high values of oxidative browningalso had high PPO specific activities by the catechol assay.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Particular aspects and methods of the present invention arenot limited to the descriptions of the specific embodiments presented,but rather the descriptions and examples should be viewed in terms ofthe claims that follow and their equivalents. While some of the examplesand descriptions above include some conclusions about the way theinvention may function, the inventors do not intend to be bound by thoseconclusions and functions, but put them forth only as possibleexplanations.

It is to be further understood that the specific embodiments of thepresent invention as set forth are not intended as being exhaustive orlimiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and scope ofthe following claims.

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1. A method of reducing polyphenol oxidase activity in a plantcomprising: transforming the plant with a transgene encoding a dsRNA,said transgene comprising a first sequence as set forth in SEQ ID NO: 1and a second sequence as set forth in SEQ ID NO: 2, wherein saidtransgene is expressed in said plant.
 2. The method of claim 1 whereinsaid plant is a potato plant.
 3. A plant comprising a transgene encodinga dsRNA, said transgene comprising a first sequence as set forth in SEQID NO: 1 and a second sequence as set forth in SEQ ID NO: 2, whereinsaid transgene is expressed in said plant.
 4. Progeny or seed of theplant of claim 3, wherein said progeny or seed comprise said transgene.5. The plant of claim 3, wherein said plant is a potato plant. 6.Progeny of the plant of claim 5, wherein said progeny comprise saidtransgene.
 7. A method for reducing susceptibility of a plant to diseasecomprising: transforming the plant with a transgene encoding a dsRNA,said transgene comprising a first sequence e as set forth in SEQ ID NO:1 and a second sequence as set forth in SEQ ID NO: 2, wherein saidtransgene is expressed in said plant.
 8. A method for reducingsusceptibility of potato to bruising comprising: transforming the plantwith a transgene encoding a dsRNA, said transgene comprising a firstsequence e as set forth in SEQ ID NO: 1 and a second sequence as setforth in SEQ ID NO: 2, wherein said transgene is expressed in saidplant.